The thymus is a bi-lobed lymphatic organ located in the anterior portion of the ventral thoracic cavity, just behind the sternum. Because the thymus is the site of development of T lymphocytes (T cells), it is frequently targeted in research studies that involve the immune system. Furthermore, the rapid expansion of transgenic and gene-targeted mouse models of immune disorders has enabled a concomitant increase in the number of studies of T-cell development. Such studies may require the administration of intrathymic injections, which have traditionally been done using a surgical approach. Surgical manipulation can result in pain or distress to the animal, which may affect the immune system, potentially confounding experimental results. Here, the authors describe a nonsurgical, ultrasound-guided approach for intrathymic injection in the mouse that results in negligible distress to the animal.